Lawrence, Christopher B.

Abstract Eosinophils produce and release various proinflammatory mediators and also show immunomodulatory and tissue remodeling functions; thus, eosinophils may be involved in the pathophysiology of asthma and other eosinophilic disorders as well as host defense. Several major questions still remain. For example, how do human eosinophils become activated in diseased tissues or at the site of an immune response? What types of host immunity might potentially involve eosinophils? Herein, we found that human eosinophils react vigorously to a common environmental fungus, Alternaria alternata, which is implicated in the development and/or exacerbation of human asthma. Eosinophils release their cytotoxic granule proteins, such as eosinophil-derived neurotoxin and major basic protein, into the extracellular milieu and onto the surface of fungal organisms and kill the fungus in a contact-dependent manner. Eosinophils use their versatile β2 integrin molecule, CD11b, to adhere to a major cell wall component, β-glucan, but eosinophils do not express other common fungal receptors, such as dectin-1 and lactosylceramide. The I-domain of CD11b is distinctively involved in the eosinophils’ interaction with β-glucan. Eosinophils do not react with another fungal cell wall component, chitin. Because human eosinophils respond to and kill certain fungal organisms, our findings identify a previously unrecognized innate immune function for eosinophils. This immune response by eosinophils may benefit the host, but, in turn, it may also play a role in the development and/or exacerbation of eosinophil-related allergic human diseases, such as asthma.

Abstract Eosinophils are multifunctional leukocytes implicated in the pathogenesis of asthma and in immunity to certain organisms. Associations between exposure to an environmental fungus, such as Alternaria, and asthma have been recognized clinically. Protease-activated receptors (PARs) are G protein-coupled receptors that are cleaved and activated by serine proteases, but their roles in innate immunity remain unknown. We previously found that human eosinophils respond vigorously to Alternaria organisms and to the secretory product(s) of Alternaria with eosinophils releasing their proinflammatory mediators. In this study, we investigated the roles of protease(s) produced by Alternaria and of PARs expressed on eosinophils in their immune responses against fungal organisms. We found that Alternaria alternata produces aspartate protease(s) and that human peripheral blood eosinophils degranulate in response to the cell-free extract of A. alternata. Eosinophils showed an increased intracellular calcium concentration in response to Alternaria that was desensitized by peptide and protease ligands for PAR-2 and inhibited by a PAR-2 antagonistic peptide. Alternaria-derived aspartate protease(s) cleaved PAR-2 to expose neo-ligands; these neo-ligands activated eosinophil degranulation in the absence of proteases. Finally, treatment of Alternaria extract with aspartate protease inhibitors, which are conventionally used for HIV-1 and other microbes, attenuated the eosinophils’ responses to Alternaria. Thus, fungal aspartate protease and eosinophil PAR-2 appear critical for the eosinophils’ innate immune response to certain fungi, suggesting a novel mechanism for pathologic inflammation in asthma and for host-pathogen interaction.

Abstract Thymic stromal lymphopoietin (TSLP) is produced by epithelial cells and triggers dendritic cell-mediated Th2-type inflammation. Although TSLP is up-regulated in epithelium of patients with asthma, the factors that control TSLP production have not been studied extensively. Because mouse models suggest roles for protease(s) in Th2-type immune responses, we hypothesized that proteases from airborne allergens may induce TSLP production in a human airway epithelial cell line, BEAS-2B. TSLP mRNA and protein were induced when BEAS-2B cells were exposed to prototypic proteases, namely, trypsin and papain. TSLP induction by trypsin required intact protease activity and also a protease-sensing G protein-coupled receptor, protease-activated receptor (PAR)-2; TSLP induction by papain was partially dependent on PAR-2. In humans, exposure to ubiquitous airborne fungi, such as Alternaria, is implicated in the development and exacerbation of asthma. When BEAS-2B cells or normal human bronchial epithelial cells were exposed to Alternaria extract, TSLP was potently induced. The TSLP-inducing activity of Alternaria was partially blocked by treating the extract with a cysteine protease inhibitor, E-64, or by infecting BEAS-2B cells with small interfering RNA for PAR-2. Protease-induced TSLP production by BEAS-2B cells was enhanced synergistically by IL-4 and abolished by IFN-γ. These findings demonstrate that TSLP expression is induced in airway epithelial cells by exposure to allergen-derived proteases and that PAR-2 is involved in the process. By promoting TSLP production in the airways, proteases associated with airborne allergens may facilitate the development and/or exacerbation of Th2-type airway inflammation, particularly in allergic individuals.

Abstract Asthma is thought to result from dysregulated Th2-like airway inflammatory responses to the environment. Although the etiology of asthma is not fully understood in humans, clinical and epidemiological evidence suggest a potential link between exposure to environmental fungi, such as Alternaria, and development and/or exacerbation of asthma. The goal of this project was to investigate the mechanisms of airway Th2 responses by using Alternaria as a clinically relevant model for environmental exposure. Airway exposure of naive animals to an experimental Ag, OVA, or a common allergen, short ragweed pollen, induced no or minimal immune responses to these Ags. In contrast, mice developed strong Th2-like immune responses when they were exposed to these Ags in the presence of Alternaria extract. Extracts of other fungi, such as Aspergillus and Candida, showed similar Th2 adjuvant effects, albeit not as potently. Alternaria stimulated bone marrow-derived dendritic cells (DCs) to express MHC class II and costimulatory molecules, including OX40 ligand, in vitro. Importantly, Alternaria inhibited IL-12 production by activated DCs, and DCs exposed to Alternaria enhanced Th2 polarization of CD4+ T cells. Furthermore, adoptive airway transfer of DCs, which had been pulsed with OVA in the presence of Alternaria, showed that the recipient mice had enhanced IgE Ab production and Th2-like airway responses to OVA. Thus, the asthma-related environmental fungus Alternaria produces potent Th2-like adjuvant effects in the airways. Such immunogenic properties of certain environmental fungi may explain their strong relationships with human asthma and allergic diseases.